Chapter 22: Structural and signaling functions of integrins

Journal Pre-proof Chapter 22: Structural and signaling functions of integrins

Yasmin A. Kadry, David A. Calderwood PII:

S0005-2736(20)30032-8

DOI:

https://doi.org/10.1016/j.bbamem.2020.183206

Reference:

BBAMEM 183206

To appear in:

BBA - Biomembranes

Received date:

16 October 2019

Revised date:

21 January 2020

Accepted date:

22 January 2020

Please cite this article as: Y.A. Kadry and D.A. Calderwood, Chapter 22: Structural and signaling functions of integrins, BBA - Biomembranes(2020), https://doi.org/10.1016/ j.bbamem.2020.183206

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© 2020 Published by Elsevier.

Journal Pre-proof

Chapter 22: Structural and signaling functions of integrins Yasmin A. Kadry1 and David A. Calderwood1,2 1

Department of Pharmacology and the 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520 *Corresponding author: David A. Calderwood ([email protected]) Introduction ......................................................................................................... 2

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Integrin structure and ligand specificity ........................................................... 3 2.1 Extracellular Domains (Ectodomains) ............................................................ 5 2.2 Transmembrane helices .................................................................................. 6 2.3 Cytoplasmic tails.............................................................................................. 8

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Integrin activation and signaling ....................................................................... 9 3.1 Intracellular adaptor binding and conformational activation ..................... 10 3.1.1 Talins in integrin activation ........................................................................ 10 3.1.2 Kindlins in integrin activation ..................................................................... 12 3.1.3 Thermodynamics of ectodomain extension ............................................... 14 3.2 Extracellular ligand engagement and Integrin clustering........................... 14 3.3 Focal adhesion assembly and the integrin adhesome ............................... 15 3.3.1 Integrin-linked kinase (ILK) ........................................................................ 15 3.3.2 Paxillin ....................................................................................................... 16 3.4 Integrin inactivation and trafficking ............................................................. 18

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Epithelial integrins ............................................................................................ 18 Hemidesmosomes and focal adhesions ...................................................... 19 Cell adhesion during mitosis ........................................................................ 20 The role of integrins in establishing epithelial cell polarity ....................... 20

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Concluding Remarks......................................................................................... 21

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Abstract

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The integrin family of transmembrane adhesion receptors is essential for sensing and adhering to the extracellular environment. Integrins are heterodimers composed of non-covalently associated α and β subunits that engage extracellular matrix proteins and couple to intracellular signaling and cytoskeletal complexes. Humans have 24 different integrin heterodimers with differing ligand binding specificities and non-redundant functions. Complex structural rearrangements control the ability of integrins to engage ligands and to activate diverse downstream signaling networks, modulating cell adhesion and dynamics, processes which are crucial for metazoan life and development. Here we review the structural and signaling functions of integrins focusing on recent advances which have enhanced our understanding of how integrins are activated and regulated, and the cytoplasmic signaling networks downstream of integrins.

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Integrin; Talin; Kindlin; Focal adhesion; Cytoskeleton

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1. Introduction

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Interactions between cells and the extracellular matrix (ECM) are a defining and essential feature of multicellular life. The integrin family of type I transmembrane adhesion receptors are primarily responsible for mediating cell-matrix attachments, but integrins can also be involved in cell-cell attachments. By coupling the extracellular environment to cytoplasmic signaling inside of the cell, integrins support cell anchorage and hence tissue and organismal architecture. Loss or dysfunction of integrins or integrin-associated proteins perturbs tissue development (e.g. in epidermolysis bullosa or Kindler syndrome) and integrin function is altered in cancer, musculoskeletal, cardiovascular and inflammatory diseases [1–4]. Indeed, integrin inhibitors are used in cardiovascular and auto-immune disease and are in trials for other indications [5] .

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Integrins are heterodimers of α and β subunits, each of which is a type I transmembrane protein with large multidomain extracellular portions, a single-pass transmembrane region and a generally short cytoplasmic tail. Humans express 18 α and 8 β subunits that combine to form 24 different integrin heterodimers exhibiting overlapping but non-redundant functions, with ligand and signaling specificity dictated by the particular combination of the α and β subunit in each heterodimer [6]. This allows integrins to engage a diverse range of extracellular ligands, including secreted ECM proteins such as fibronectin, laminin and collagen, and Ig-superfamily cell-surface receptors such as intercellular adhesion molecule-1 (ICAM-1) and vascular celladhesion molecule-1 (VCAM-1) [7]. Integrins function by bi-directionally transducing biochemical signals and mechanical force across the plasma membrane. This requires engagement of extracellular ligands by the integrin extracellular domains and of intracellular signaling and cytoskeletal proteins by the integrin cytoplasmic tails. The specific repertoire of binding proteins and how the interactions are mechanically regulated dictates integrin signaling and regulates the link to the actin cytoskeleton [8]. Notably, engagement of the integrin cytoplasmic domains with specific intracellular binding

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Journal Pre-proof partners can induce integrin clustering and trigger conformational rearrangements of the integrin transmembrane and extracellular domains that increase the affinity of integrin extracellular domains for ligand (integrin activation) [9]. Intracellular signals thus regulate cell adhesion through allosteric changes in integrin extracellular domains. Likewise, extracellular ligand binding can also drive changes in cytoplasmic domain interactions facilitating integrin signaling [7].

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2. Integrin structure and ligand specificity

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Integrins and their roles in cell adhesion, migration and signaling in health and disease have recently been extensively reviewed elsewhere [10–13], therefore here we summarize the fundamentals of integrin structure, activation and regulation that are applicable to the majority of cell lineages and focus on more recent advances in conformational regulation and thermodynamics of integrin activation, and on a subset of the key protein-protein interactions within the integrin signaling network. Finally, we discuss functions of integrins that are relevant to epithelial cells.

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Integrins are conserved throughout the metazoan kingdom (multicellular animals), presumably reflecting the importance of cell adhesion systems for multicellularity [14,15]. Notably, from genome sequencing efforts, it is now recognized that integrins and integrin-associated proteins can also be found in some unicellular relatives of the Metazoa, suggesting that adhesion systems were already present in the single-celled or colonial ancestors of the Metazoa, although their roles there are unknown [14,15]. In vertebrates, integrins are expressed in nearly all cell types, but cell-lineage restricted expression patterns of specific α and β subunits control the abundance of different integrin heterodimers found on each cell[7]. In addition, multiple splice variants of some subunits have been identified, which adds an additional layer of complexity to ligand binding and signaling specificity [16]. Despite these complications, integrin heterodimers share a conserved architecture consisting of a large extracellular (ectodomain) region, a transmembrane region, and generally short intracellular cytoplasmic tails (Fig. 1A,B). Over the past 25 years there have been major advances in our understanding of the structure and dynamics of integrins and their interactions with extracellular and intracellular ligands – these findings have been extensively reviewed elsewhere [17–21] and are briefly summarized below.

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Journal Pre-proof Figure 1: Integrin domain architecture and conformational states. (A,B) Schematic of the domain architecture of an αA domain-containing (A) and non-αA domain containing (B) integrin heterodimer in the active (extended-open) state. Metal-ion binding sites (MIDAS, ADMIDAS, and SyMBS) are indicated. (C) Cartoon depiction of the conformational transitions of the integrin heterodimer from a bent-closed (BC) to an extended-closed (EC) and finally to an extended-open (EO) conformation. 2.1 Extracellular Domains (Ectodomains)

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The  and  subunits of integrins are unrelated in sequence but a stable non-covalent association between their multidomain extracellular portions is required for normal traffic to the cell surface and for regulated ligand binding [6,22]. The amino acid sequence of  subunits, and of  subunits, is conserved across species but a subset of vertebrate integrin  subunits (half of the 18 mammalian α subunits) contain an additional ~200 amino acid insertion in the extracellular portion (Fig. 1A). The inserted region, known as an αI domain or an αA domain (due to its similarity to the A-domain of von Willebrand factor), is found in integrins that bind collagen and in some laminin-binding integrins, where it is the major site of ligand binding [23,24]. The Adomain was the first integrin domain to be structurally characterized at high resolution and this revealed the basis of direct ligand binding to A-domains and its dependence on divalent cation coordination at the A-domain “metal-ion dependent adhesion site” (MIDAS) [25,26] (Fig. 1A). The four αA-domain containing collagen binding integrins (α1β1, α2β1, α10β1, and α11β1) recognize a “G-F-O-G-E-R” motif, in which the key acidic Glu coordinates with a cation that is bound to the αA domain [27]. The αA-domain containing family also includes the β2 family of integrins (αDβ2, αLβ2, αMβ2, αXβ2), which interact with ligands such as ICAM through recognition of a consensus L/I-D/E-V/S/T-P/S sequence where the acidic residue again plays a key role in binding through coordination of the divalent cation [28]. The last member of the αAdomain containing integrin family, αEβ7, has been found to be important for lymphocyte adhesion to epithelial cells, likely by binding to an exposed glutamate residue in E-cadherin through a cation in the αA domain [29].

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Although crystal structures of the integrin A-ligand complexes revealed the basis of ligand binding for a subset of integrins [25,26], the first detailed structural insights into intact integrin ectodomains were garnered in a series of crystallography and electron microscopy studies performed from the early 2000s [30–32]. Initial crystal structures of the αvβ3 ectodomain [30,31] revealed that the ectodomain of each α and β subunit consists of multiple domains that are flexibly linked. Each α chain consists of two calf domains and a thigh domain that adopt IgG-like folds, and a β-propeller domain (Fig. 1A,B) [30]. In integrins which have an αA domain it is inserted into a loop in the β-propeller domain [21]. The ectodomain of the β-subunit consists of a βA domain (also known as a βI domain) that is analogous to the αA domain, a hybrid domain with a β-sandwich architecture, a PSI-domain with an α-β fold, four cysteine-rich EGF modules, and a β-tail domain adopting an α+β fold [30] (Fig. 1A,B). In integrins lacking an αA domain, interactions between the β-propeller in the α subunit and βA domain in the β subunit form the ligand binding “head” of the heterodimer (Fig. 1B) [21]. The head coordinates interactions with charged residues in ligands in a divalent cation-dependent manner (Fig. 1B) [21]. These cations are found in three sites within the βA domain: a ‘metal-ion

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Journal Pre-proof dependent adhesion site’ (MIDAS), an adjacent MIDAS (ADMIDAS), and a third synergistic metal-ion binding site (SyMBS) (Fig. 1B) [21]. Eight of the 24 integrin heterodimers (αIIbβ3, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1) recognize an ‘R-G-D’ motif in the ligand via interactions between the charged residues in the ligand and coordinating residues and cations in the ligand-binding head [7]. Three other non-αA domain containing integrins (α4β1, α4β7, and α9β1) recognize an acidic consensus sequence, defined as L/I-D/E-V/S/T-P/S [7]. For a small subset of laminin-binding integrins (α3β1,α6β1,α7β1, α6β4), limited information is available on the binding mode of the laminin-heterotrimer to the integrin heterodimer, but structural studies on laminin-111and laminin-511 suggests that a surface-exposed glutamic acid in laminin 1 chain may interact with the MIDAS in the β-subunit [33,34].

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A key feature of integrin ectodomains is their ability to adopt a range of conformations with distinct affinity for ligands. The first integrin ectodomain structures [30,31] led to the surprising finding that the ligand-binding head piece folded back against the  and  subunit legs resulting in a highly bent structure (Fig. 1C). This, together with electron microscopy studies, a range of biophysical measurements and further structural studies [35,36] ultimately led to the now generally accepted model where integrins can adopt at least three main conformations. These are a low-affinity bent-closed (BC) conformation, an intermediate-affinity extended-closed (EC) conformation, and a high-affinity extended-open (EO) conformation where the head piece is open with the hybrid domain swung out stabilizing the high-affinity conformation of the A domain and propagating conformational change along the integrin legs to the transmembrane and cytoplasmic regions (Fig. 1C). As discussed in later sections, the conversion from low affinity to high affinity conformations (integrin activation) is a tightly regulated process.

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2.2 Transmembrane helices

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The single-pass transmembrane helical domains (TMD) of the α and β subunit (the α-TMD and β-TMD, respectively) non-covalently associate within the plasma membrane (Fig. 1A,B) [37]. The α-TMD and β-TMD serve as a mediary between the ectodomains and intracellular cytoplasmic tails, and are essential for transmission of the long-range conformational changes that propagate through the integrin heterodimer during activation. The importance of the association of the α-TMD and β-TMD for integrin function was first gathered from mutagenic studies, which demonstrated that mutations which disturb the non-covalent association between the α and β transmembrane helices can activate integrins [37–39]. These studies engendered the idea that the activation state of an integrin directly correlates with the dissociation state of the αTMD and β-TMD, whereby association of the α and β transmembrane helices is observed in the inactive state of the integrin heterodimer, and disruption of the association between the transmembrane helices, triggered by interaction(s) with cytoplasmic proteins, promotes integrin activation [40]. This model was supported by the NMR structure of the αIIbβ3 transmembrane complex in phospholipid bicelles, which revealed that the transmembrane helices of the α and β subunit interact at two interfaces which are specified by a 25 degree tilt of the transmembrane helix of the β subunit [41]. The first interface, termed the ‘outer membrane clasp’ (OMC), is composed of a helical packing interaction at the N-terminal region of the TMD that is dependent on a ‘GxxxG’ motif in the α subunit and G703 in the β subunit (Fig. 2A) [40,41]. The second interface is known as the ‘inner membrane clasp’ (IMC), and is formed by a break in the α

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subunit transmembrane helix introduced by a ‘GFFKR’ motif (Fig. 2A). The break in the α subunit transmembrane helix turns the α subunit towards the β3 subunit, facilitating hydrophobic stacking interactions between F992/F993 of the α subunit and W715/I719 in the β subunit, as well as a salt bridge between R995 in the α subunit and D723 in the β subunit (Fig. 2A) [40,41]. In order for integrin activation by cytoplasmic signals to occur (also known as ‘inside-out’ activation), both the OMC and IMC must be disrupted by the propagation of conformational changes across the α-TMD and β-TMD [19].

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Figure 2: The integrin α-TMD and β-TMD. Solution NMR structure of the α-TMD and β-TMD of IIb3 (PDB ID: 2K9J), with the outer membrane clasp (OMC) and inner membrane clasp (IMC) as indicated [41]. Both the OMC and IMC are important for maintaining controlled regulation of the association state of the α-TMD and β-TMD and consequently integrin activation, and the formation of the OMC and IMC are highly dependent on the transmembrane tilt of the β subunit transmembrane helix [41,42]. In particular, this topography is highly dependent on a conserved lysine near the IMC that ‘snorkels’, or embeds itself, into the plasma membrane through interactions with negatively charged phospholipid head groups in the plasma membrane [42]. Mutation of this lysine to alanine promotes dissociation of the α-TMD and β-TMD and induces spontaneous integrin activation [42]. The effect of this mutation can be compensated by the introduction of a proline at the midpoint of the β3 transmembrane helix, which induces a kink and subsequently re-tilts the β3 transmembrane helix and restores association between the α-TMD and β-TMD [42]. Therefore, the precise orientation of the α and β transmembrane helices is an important component of regulation of integrin activation state.

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Moreover, coupling between the OMC and IMC is important for propagating conformational changes from the cytoplasmic tails across the TMD and into the ectodomains. Introduction of a proline residue into the β3 transmembrane helix between the OMC and IMC disrupts coupling between the OMC and IMC by effectively ‘breaking’ the transmembrane helix, and prevents the transmission of conformational changes from the C-terminus of the helix to the N-terminus that occurs when activating proteins impinge upon the cytoplasmic tails [19,42]. Recent work has demonstrated that the introduction of this proline kink in two other integrins, β2 and β7, similarly decouples the IMC and OMC, and prevents the transmission of activating signals from the cytoplasm across the plasma membrane [43,44]. Thus, the coupling between the OMC and IMC in the TMD is likely to be a conserved mechanism in many integrins, especially considering the remarkable conservation of the protein sequences of each of the α and β transmembrane segments [41].

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2.3 Cytoplasmic tails

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The integrin cytoplasmic tails serve as nucleation center for protein-protein interactions between integrins and intracellular proteins [8,20] (Fig. 1A,B). Both the α and β cytoplasmic tails can bind various cytoplasmic adaptors, but known β-subunit interactors are more numerous and wellcharacterized [45–47]. The length of the β cytoplasmic tail ranges from 40-70 amino acids [48], with the exception of β4 integrin, which has an unusually long β cytoplasmic tail of approximately 1000 amino acids, and unlike most integrins, couples to the intermediate filament cytoskeleton [49]. With the exception of the 4 cytoplasmic tail, which contains a Calx- motif and four fibronectin type III domains involved in interactions with the plakins, plectin and BP230 [50–52], most short integrin  tails are largely unstructured [53–55]. However, regions with propensity to form helices or turns when packing against membrane or other proteins have been identified, and the integrin tails can form defined structures when bound to cytoplasmic partners [53,54,56–58].

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While the 8 β-tail protein sequences in humans are moderately conserved, several motifs serve as binding ‘hot spots’ for intracellular adaptor proteins (Fig. 3). The majority of β-tails contain a plasma membrane proximal ‘NP(I/L)(Y/F)’ motif and a membrane distal ‘Nxx(Y/F) motif’, as well as an intervening serine/threonine (S/T) rich motif (Fig. 3) [59]. Many intracellular adaptor proteins interact with the NP(I/L)(Y/F)/Nxx(Y/F) motifs through a canonical PTB domain (or PTB domain-like)-NP(I/L)(Y/F)/Nxx(Y/F) interaction, that may or may not require tyrosine phosphorylation on the NP(I/L)(Y/F)/Nxx(Y/F) motif [59]. In addition, phosphorylation of the S/T rich motif can serve to regulate adaptor binding either positively or negatively [60]. A few key integrin β-tail interactions are discussed in subsequent sections.

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Figure 3: Sequence alignment of -tail cytoplasmic domains. Sequence alignment of human -integrin cytoplasmic tail protein sequences. Conserved ‘NP(I/L)(Y/F)’, ‘Nxx(Y/F)’, and Serine/Threonine rich motifs are indicated. Alignment was generated using the ESPript web server, and ESPript-calculated conserved residues are colored in red and boxed in blue [61]. 4 was omitted from this alignment due to its unusually large length, and 8 was excluded due to a lack of strong conservation with the other  subunits in the alignment.

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In contrast to the  integrin tails, the α subunit cytoplasmic tails tend to be much shorter and, with the exception of a conserved membrane-proximal ‘GFFXR’ motif, are much more divergent among isoforms [62]. Binding partners of the -tails have received less attention than those of the -tail but -tail interactions are increasingly recognized for their roles in controlling integrin membrane trafficking and for inhibition of integrin activation [63,64]. In addition, an interaction between the 4 tail and Hsp90 has recently been identified and shown to be important for controlling the T-cell thermal sensory response to fever, suggesting that -tail interactions may also regulate integrin function during the immune response [65].

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3. Integrin activation and signaling The modulation of integrin affinity for extracellular ligands is tightly regulated through a multistep activation process that consists of several major stages: intracellular adaptor recruitment and conformational activation, ECM ligand engagement, clustering/focal adhesion assembly, and finally, integrin inactivation (Fig. 4). These processes have recently been extensively reviewed elsewhere [10,11,18] and are briefly summarized here. During the first stages, regulatory intracellular adaptors interact with the integrin cytoplasmic tails, triggering long-range conformational changes in the integrin heterodimer that positively modulates the affinity for extracellular ligands. The integrin heterodimer then engages with extracellular ligands and connects to the force-transducing actin cytoskeleton, which together promote full activation and integrin clustering to form complex cell-matrix attachments known as focal adhesions [66,67]. This clustering, and the application of externally applied or internally actomyosin-generated tension triggers the sequestration of additional intracellular binding partners and subsequent downstream signal transduction [8,19]. Integrin signaling ultimately culminates in integrin inactivation, which is driven by processes such as endocytic recycling [68]. In non-adherent cells, integrin activation regulates cell adhesion in processes such as leukocyte migration into

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tissue during the immune response [69]; in constitutively adherent cells, this process regulates the modulation of cell-matrix and cell-cell adhesion that are relevant in processes such as wound healing[70].

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Figure 4: The integrin activation process. A partial overview of the integrin activation process. First, intracellular adaptor recruitment drives conformational activation and the transition from the bent-closed to extended-open state. Following this, integrin clustering and focal adhesion assembly begins. Finally, focal adhesion disassembly/integrin inactivation is promoted by the recruitment of endocytic/inhibitory adaptors to the cytoplasmic tails.

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3.1 Intracellular adaptor binding and conformational activation

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Integrins are able to interface with various cytoplasmic adaptors through direct and indirect interactions between these adaptors and sequence motifs in the cytoplasmic tail of the β-subunit [8,20,71]. These interactions regulate the activation state of the integrin, which is important for processes such as cell migration and wound healing. Typically, the two highly-conserved ‘NP(I/L)(Y/F)’/’Nxx(Y/F)’ motifs in the cytoplasmic tail of the β-subunits bind proteins containing phosphotyrosine binding (PTB) or PTB-like domains [59]. These proteins can be of an activating or inhibitory nature. A comprehensive discussion of all the known integrin tail binding proteins and their effects on integrin activation and signaling is beyond the scope of this review. Instead, we briefly discuss two of the most well-studied cytoplasmic interactors, the integrin activators talin and kindlin. 3.1.1

Talins in integrin activation

Talin is arguably the most important cytoplasmic binding partner of integrins and the first wellcharacterized integrin activator [72–74]. Talin is central to integrin activation, provides a direct link between integrins and the actin cytoskeleton, is mechanically regulated, and serves to recruit additional signaling and cytoskeletal proteins to ligand-bound integrins; as such, talin is integral

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to integrin function and is the subject of numerous recent reviews [11,75–78]. Talin proteins are conserved throughout Metazoans and can even be found in some unicellular relatives of the Metazoa [14,15]. Notably, the talin-binding site on the β-cytoplasmic tail is conserved in premetazoan organisms, suggesting an evolutionarily conserved function of talin binding [79]. Mammals express two closely related but differentially expressed talin isoforms, with overlapping but non-redundant functions; Talin-1 is the most ubiquitously expressed and wellstudied isoform, while Talin-2 is more restricted [9]. Talin loss in mouse, Drosophila, or C. elegans [80–83] produces severe adhesion phenotypes consistent with the central role of talin in integrin function. Talins are large mechanosensitive proteins consisting of an N-terminal ‘talin head’ composed of an atypical 4.1-ezrin-radixin-moesin (FERM) domain and a C-terminal rod domain composed of 13 helical bundles and a C-terminal dimerization domain (although the extent and significance of dimerization remains unclear [84]). Talin binds directly to β-integrin cytoplasmic tails through one of two integrin binding sites: one located in the talin head domain (IBS1), and another located in the rod domain (IBS2) [73,85]. The binding of the talin head is indispensable for inside-out integrin activation and structural studies have revealed that the F3 subdomain of the talin head interacts with a plasma membrane-proximal ‘NP(I/L)(Y/F)’ through a modified PTB-NPxY interaction [57]. Talin binding destabilizes the association of the  and  tails close to the membrane and disrupts intramembrane interactions between the transmembrane helices of the integrin α and β subunits, leading to conformational changes in the heterodimer that promote an active state [44,86,87]. Interactions between the talin head and the acidic phospholipid PIP2 also contribute to the induction of these conformational changes, presumably by spatially orienting the talin head to promote integrin activation [88].

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In addition to binding and conformationally activating integrins, talin connects integrins to the cytoskeleton and to downstream signaling pathways through direct and indirect interactions with other proteins, and studies in Drosophila suggest that talin can modulate adhesion via a range of alternative mechanisms [89]. Notably, talin binds directly to F-actin [90], and two actin-binding sites within the talin rod (referred to as ABS2 and ABS3) play distinct roles in the assembly of focal adhesions [91]. Talin also binds to the actin-binding protein vinculin [75,92], providing a direct link from the ECM, via integrins and talin, to the actin cytoskeleton. This connection permits transmission of mechanical force and distribution of forces through the cytoskeleton, and mechanical stretching of talin has been observed in cells and has been shown to favor vinculin binding [93–96]. Force applied to talin exposes cryptic vinculin binding sites within the helical bundles that make up the talin rod, each bundle unfolding at a distinct force profile via a process that is reversible [93]. Talin binding to numerous other partners, including deleted in liver cancer-1 (DLC1), FAK, layilin, KANK family proteins (KANK1-4), the Ras-family small GTPase Rap1, the Rap1 effector RIAM, and membrane lipids has been reported and in some cases these interactions have been structurally characterized [97–103]. Among these, Rap1 and RIAM1 have recently received a great deal of attention for their roles in recruiting talin to the membrane and facilitating conformational activation of talin allowing subsequent engagement and activation of integrins. In contrast to vinculin, the talin-RIAM interaction is inhibited by talin rod unfolding induced by mechanical force, suggesting that force may act as a molecular switch to regulate vinculin/RIAM interactions with talin [104]. While RIAM-mediated talin recruitment and activation is important in certain settings [105–110], it is now apparent that the talin head contains two direct Rap1 binding sites (in the F0 and F1 domains) and that these interactions, along with direct talin head-membrane interactions, are important for talin regulation of integrin

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There is abundant in vitro and in vivo evidence that intramolecular interactions between the FERM domain and rod domain maintain talin in an autoinhibited state, and that regulation of autoinhibition modulates cell-ECM adhesion and migration [84,116–120] . The recent cryo-EM structure of intact autoinhibited talin1 confirms the autoinhibition, revealing the nature of the many intramolecular interactions involved in holding talin inactive [84]. Notably, in this structure the F0 and F1 domains are exposed and presumably competent to bind Rap1, potentially allowing talin recruitment to the membrane and favoring talin activation. Autoinhibited talin does however perturb normal talin head interactions with integrin, and some talin-rod interactions and can be released following binding of talin to vinculin, RIAM and potentially KANK proteins [107,121,122].

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Finally, by binding directly to KANKs, a scaffold protein that associates with the cortical microtubule stabilizing complex [101], talin controls microtubule tip recruitment to focal adhesions, influencing adhesion turnover and remodeling through delivery of matrix metalloproteases and modulation of Rho family GTPase signaling [101,122–124]. The contribution of focal adhesion-associated microtubules to integrin signaling has become increasingly appreciated and appears to be especially important for control of focal adhesion dynamics and turnover [125]. The interaction of KANKs with the talin rod domain also influences talin activation and hence integrin activation [101,122]. Thus, talin is involved at multiple steps throughout the lifecycle of integrin-mediated adhesions, from initial integrin activation, through linkage to actin and force transmission, protein scaffolding and adhesion turnover.

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3.1.2 Kindlins in integrin activation

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During the initial studies on integrin activation in the late 1990s and early 2000s, talin was thought to be the sole mediator of integrin activation [72–74]. Subsequent knockdown, knockout, and overexpression studies revealed that the kindlins are also essential for integrin activation and signaling [126–132]. At least one kindlin-encoding gene can be found in all metazoan lineages, but phylogenetic analysis suggests that kindlins appeared later in evolution, likely from a duplication and later modification of the talin FERM domain [133]. There are three mammalian kindlin isoforms, each of which are differentially expressed and possess overlapping but nonredundant functions; kindlin-1 is found primarily in epithelial cells, kindlin-2 is nearly ubiquitously expressed, and kindlin-3 is expressed primarily in cells of the hematopoietic lineage and possibly in some endothelial cells [134,135]. Consistent with this expression pattern, loss of kindlin-1 results in Kindler syndrome which is associated with skin blistering and photosensitivity while loss of kindlin-3 causes leukocyte adhesion deficiency type III, with both diseases linked to defects in integrin activation and cell adhesion [136–139]. Kindlin proteins are formed from an atypical FERM domain [140]. Like the talin head, the kindlin FERM domain contains an F0 subdomain, in addition to the canonical FERM domain F1, F2 and F3 subdomains. Kindlins also have a large insertion in the F1 subdomain, and unlike the talin FERM, a pleckstrin homology (PH) domain nestled within the F2 subdomain [140,141]. 12

Journal Pre-proof The kindlin F3 subdomain directly interacts with the membrane distal ‘Nxx(Y/F)’ on the βcytoplasmic tail, a site that is distinct from the talin binding region [126,128,132,140]. Mutagenesis studies and the recently reported crystal structures of a truncated form of kindlin-2 bound to the β1 and β3 cytoplasmic tails has revealed that specificity for the membrane distal ‘Nxx(Y/F)’ motif is dictated by interactions between the kindlin F3 subdomain and threonine residues N-terminal to the ‘Nxx(Y/F)’ motif on the integrin tail [129,140,142].

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The exact role of kindlins in integrin activation is still incompletely understood but appears to be quite complex, and may be cell-type and integrin heterodimer dependent. Multiple overexpression [128,129,143] and knockdown/knockout [126,129,144,145] studies in mammalian cells, make it clear that kindlins are important for the activation of integrins, and this has been shown to be dependent largely on direct interactions with integrins [128,146]. Other studies, however, have reported that kindlins can be suppressive of integrin activation; when overexpressed in CHO cells, for example, kindlins have been demonstrated to suppress 51 integrin activation rather promoting activation [128]. Recent work, however, has demonstrated that kindlins can bind to and recruit the integrin inactivator SHARPIN to 51, providing a mechanism for the suppressive effects of kindlins on integrin activation [147]. A multitude of evidence also implicates other kindlin interactors, such as paxillin [148,149], integrin-linked kinase (ILK) [146,150], and actin [151] as contributing to the role of kindlins in integrin activation and/or subsequent adhesion signaling. While the means by which kindlin is regulated to enhance or suppress integrin activation and signaling is currently unclear, the contributions of some of these binding partners will be discussed in later sections.

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Currently, the mechanistic details regarding how direct interactions between kindlin and the βcytoplasmic tail promote integrin activation is unclear. The current prevailing model is that the major contribution of kindlins to integrin activation stems from cooperation with talin to enhance talin-mediated integrin activation, as overexpression studies suggest that talin-mediated activation of IIb3 integrin is enhanced when talin FERM is co-overexpressed with kindlins, and impaired when co-overexpressed with mutants of kindlin that are impaired in integrin binding; moreover, these studies report that kindlins alone have minimal effect on IIb3 activation in an overexpression system [128]. Although the mechanistic nature of this synergy is not clear, studies on IIb3 suggest that neither talin nor kindlin influences the binding or recruitment of one another to integrins [152]. Recent work has suggested that kindlins may cluster integrins to increase ligand binding avidity, and that this underlie the function of kindlin in integrin activation (discussed in more detail in section 3.2) although the role of kindlins in integrin clustering is unclear, and it still uncertain if this observation extends to other integrin heterodimers other than IIb3 [140,143]. It is currently unclear if kindlins, like talin, can directly induce conformational changes in the TMD of the integrin heterodimer to promote integrin activation [142]. Molecular dynamics simulations using a recently solved crystal structure of a kindlin-2 lacking the PH domain and a portion of the F1 subdomain suggests that this is not the case, and that rather, kindlins assist talin to fully destabilize an inhibitory salt bridge interaction between the transmembrane helices [153]. However, even in the presence of normal levels of talin, the kindlins are still clearly important for integrin activation in a multitude of cell lineages, as loss of endogenous kindlin by knockdown or knockout leads to defects in integrin activation in mouse fibroblasts and other cell

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3.1.3 Thermodynamics of ectodomain extension

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Recent structural studies on α5β1 integrin using negative-stain electron microscopy and conformational-stabilizing antibodies have shown that the ectodomain of α5β1 transitions between three states during conformational activation: bent-closed (BC), extended-closed (EC), and extended-open (EO) (Fig. 4) [154]. The BC state is characterized by the close proximity of the ligand-binding head to the lower legs of the heterodimer, while in the EC and EO states, the ligand-binding head is elevated above the lower legs [155]. These conformational states are defined by distinct free energies, and the transition from the BC to EC and EO during integrin activation requires cellular energy input [155]. The EO state is characterized by an increased affinity for extracellular ligands, which has been measured to be 5,000-fold greater for α5β1 in K562 cells in the EO state than the BC and EC states [155]. The energy input for these conformational changes may originate from forces generated by movement of actin filaments in the cell, to which the integrins are connected by cytoplasmic binding partners like talin [66,154]. This is likely to be a general mechanism, but further thermodynamic studies on other integrin subunits, especially in the context of constitutively adherent cells, are likely to shed more light on the subject.

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Overall, the relative contributions of inside-out integrin activation and intrinsic conformational equilibrium in the regulation of ectodomain extension and whether inside-out activation (cytoskeletal force)is required in all circumstances has been widely contested. Negative stain-EM studies on the complete ectodomain of αIIbβ3 proposed that lateral force on the β3 subunit, such as that which would occur upon attachment of the β3 subunit to moving actin filaments, stabilizes the open conformation of the ectodomain [32]. Measurements of conformational equilibria of α5β1 on the surface of K562 cells have also demonstrated that a cellular energy input of ~4kcal/mol is required for the extension of the ectodomain and to stabilize headpiece opening, suggesting that input from cytoskeletal force is required [155]. Follow-up quantitative modeling supports this idea, concluding that the large-scale conformational changes that occur during integrin activation and the ultrasensitive regulation of this process requires cytoplasmic adaptor binding and cytoskeletal force [66]. However, while recent evidence increasingly suggests that input of cytoskeletal force is required for stabilization of the open, high-affinity integrin ectodomain conformation, earlier experiments showing talin-mediated activation of purified IIb3 integrins in nanodiscs in the absence of cellular forces argue that it may not always be required [156]. It is yet to be determined whether this apparent discrepancy reflects differences in methodology used to assess activation, or variation between heterodimers and/or cell type [11].

3.2 Extracellular ligand engagement and Integrin clustering After initial talin and kindlin stimulated integrin activation, integrin heterodimers engage with ECM proteins (Fig. 4), and form nascent clusters of multiple ligand-bound integrins that may grow and expand as the adhesion matures under applied forces [10,20]. The spatio-temporal

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dynamics of the integrin activation and clustering processes have yet to be elucidated, but recent work using super-resolution imaging has suggested that there can exist a large degree of conformational heterogeneity within integrin adhesions, including distinct sub-populations of both active and inactive integrins [157]. How these clusters are promoted and stabilized is currently quite controversial. One model for clustering implicates the glycocalyx in driving mechanical coupling induced by ligand binding to promotes integrin clustering [158]. Other models suggest that clustering of the extracellular ligands themselves, which can be multivalent in nature, may promote integrin clustering and subsequent adhesion stability [159]. Recently, attention has also focused on the idea that scaffolding proteins recruited to the integrins, directly or indirectly, may stabilize integrin clusters through connections to the F-actin cytoskeleton. There are numerous potential integrin tail binding proteins that could contribute to clustering but the idea of kindlins as potential mediators of integrin clustering has recently received more attention [160]. It has been reported that depletion of endogenous kindlin-2 hinders the ability of αIIbβ3 to bind multivalent fibronectin fragments, but not a monomeric fragment, suggesting that kindlins alter the avidity, rather than affinity, of αIIbβ3 for fibronectin [143]. New structural information has suggested that purified kindlins lacking the PH domain and a portion of the F1 subdomain can dimerize, albeit on an extremely slow time scale, providing a potential mechanism for clustering of integrins that may underlie the role of kindlins in integrin activation [140]. Kindlins also interact with several downstream partners that have been shown to be important for integrin-related functions, such as ILK [161,162] and paxillin [148,149], which may also play a role in the putative ability of kindlins to dimerize and cluster integrins. However, it has yet to be directly demonstrated that full-length kindlins can dimerize, and whether kindlin dimerization is important for integrin clustering.

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3.3 Focal adhesion assembly and the integrin adhesome

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Following inside-out activation and extracellular ligand engagement, clustered integrins assemble a large network of cytoplasmic and cytoskeletal proteins into focal adhesions (Fig. 4). These sites of ECM adhesion and signaling provide a link to the cytoskeleton and transmit and respond to mechanical forces, and focal adhesion assembly is driven by cell adhesion and contractility [11,18]. During the adhesion maturation process, large assemblies of multi-protein complexes are recruited to these sites of integrin-mediated adhesion. Together the many integrin associated proteins, known as the “integrin adhesome” [46,47,163], help reinforce and stabilize cell adhesion to the ECM, but also serve to relay signals downstream of integrins through protein-protein interactions. Space constraints preclude a detailed description of the many adhesome proteins and their interactions and functions. Instead we briefly highlight ILK and paxillin, because recent literature that highlights their linkage to a potentially important signaling axis centered around the kindlin proteins discussed in Section 3.1.2 (Fig. 5).

3.3.1 Integrin-linked kinase (ILK) Integrin-linked kinase (ILK) was first identified in a 1996 yeast-2-hybrid screen searching for interactors with the β1 cytoplasmic tail [164]. Although initially thought to be an active kinase, ILK is now recognized as a pseudokinase, primarily acting as a hub for protein-protein

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interactions [165–167]. ILK, which is well conserved across species, is essential for viability and consists of an N-terminal Ankyrin repeat domain (ARD) and a C-terminal pseudokinase domain (pKD) [167]. In cells, ILK is found in an obligate complex with two other proteins, PINCH (which binds the ARD) and parvin (which binds the pKD) [168–170]. This complex, known as the ILK-PINCH-Parvin (IPP) complex, is important for focal adhesion assembly and may also be mechanosensitive [171]. Studies of muscle attachment sites in Drosophila suggest that the IPP complex is essential for the stabilization of integrin-actin linkages, as well as integrin-ECM adhesion in response to mechanical force [171]. Interestingly, recent studies in mammalian cells have identified actin-binding sites in both PINCH and parvin and demonstrated that the IPP complex can bundle F-actin, further supporting the role of the IPP complex in mediating integrin-actin linkages [166]. Although initially believed to interact directly with β-integrin tails, increasing evidence suggests that the recruitment of ILK to integrins at sites of adhesion is indirect and mediated through kindlins [126,161], or through interactions between parvin and paxillin [172–174]. Kindlin binds to the pKD of ILK and the complex aids both kindlin and IPP complex targeting to adhesions [146,150,161,175]. This finding provides a mechanism by which kindlins, initially recognized as integrin activators, may also link integrins to the actin cytoskeleton.

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3.3.2 Paxillin

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Since the initial identification and characterization of paxillin in the 1980s and 1990s, paxillin has emerged to be recognized as an essential component of focal adhesions [176]. Paxillin is composed of a cluster of N-terminal “Leucine-Aspartate” (LD) motifs, and four C-terminal LIM domains [177]. Paxillin does not possess any catalytic activity, and primarily serves as a scaffold to mediate protein-protein interactions in focal adhesions [177]. Paxillin is highly phosphorylated, and both serine and tyrosine phosphorylation of paxillin serves to modulate its interaction with binding partners and subsequent signal transduction [178–180]. Paxillin is one of the first components recruited to nascent focal adhesions, and can influence focal adhesion dynamics in a manner that is dependent on its phosphorylation state [181,182]. Paxillin has been shown to complex with many other focal adhesion components, including focal adhesion kinase (FAK) [183], vinculin [184], and the IPP complex via parvin [185]. Most recently, paxillin has been reported to interact directly with the F0 and/or PH subdomains of kindlin [148,149,186]. Only the kindlin F0-paxillin LIM4 interaction has been structurally characterized [186], and the functional significance of a paxillin-binding site in the PH domain is currently unclear. Kindlins have also been reported to interact with the other paxillin family members HIC-5 [149] and leupaxin [187] and mutagenic mapping suggests this interaction is mediated by a motif in the F0 subdomain that overlaps with the paxillin binding site [134,149]. This has highlighted the possibility of a IPP complex-kindlin-paxillin signaling axis directly downstream of integrins which warrants further investigation (Fig. 5) [148,149,188].

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Figure 5: The kindlin-IPP-paxillin interactome. A summary of the major interactome connections between kindlin, paxillin and the IPP complex identified to date. Kindlin has been reported to interact with the IPP complex [146,150,161], F-actin [151], and paxillin at two distinct sites in the F0 [148,149,186,188] and PH domains [148,188], although only the kindlin F0-paxillin LIM4 interaction has been verified structurally [186]. Meanwhile, interactions between the IPP complex (through parvin) and paxillin [172,173], as well as between the IPP complex and F-actin [166], have also been reported, creating a complex signaling network centered around kindlins. Moreover, the extent of the signaling network is enhanced by reported interactions between paxillin and focal adhesion kinase [183]. Double-headed arrows indicate a reported interaction between the two binding partners. 17

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3.4 Integrin inactivation and trafficking

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The NP(I/L)(Y/F)/Nxx(Y/F) motifs on the β-integrin cytoplasmic tails are conserved recognition sequences for phosphotyrosine-binding (PTB) domain proteins [59]. In addition to binding the activators talin and kindlin, several binding partners have been identified that compete with talin and kindlin on these sites to negatively regulate integrin activation [189]. ICAP-1α is a PTBdomain containing protein that binds to the membrane distal ‘Nxx(Y/F)’ motif of the β1A tail [58] and has been shown to compete with kindlin binding at this site and to inhibit talin binding at the membrane-proximal ‘NP(I/L)(Y/F)’ motif resulting in disruption of focal adhesions [59,190,191]. In addition, the filamin family of actin-branching proteins has been shown to directly bind to the β1A-tail on a binding site that overlaps with that of talin [56]. How precisely ICAP-1α and filamin are recruited to integrins, and how this recruitment is regulated spatiotemporally, has yet to be uncovered, although regulated shuttling of ICAP-1 to the nucleus may sequester it away from integrins preventing suppression of integrin activation [192] and filaminintegrin interactions can be regulated by autoinhibition [193], competition [56,194,195] or phosphorylation [196]. Notably, in addition to competing with talin and kindlin for binding to integrin, further structural studies indicate a ternary complex where filamin also engages Nterminal helices of the  and  tails stabilizing their interaction and holding the integrin in an inactive conformation [197]. Interestingly, binding of SHARPIN to the conserved membraneproximal region of the  tail also inhibits integrin activation by impairing recruitment of talin and kindlin to the  tail [64]. As for filamin, this might involve stabilization of the inhibitory - tail association, although a recent study also reported SHARPIN association with the  tail, possibly via kindlin [147]. The potential for kindlin, normally considered an integrin activator, to recruit suppressors of integrin activation is interesting as it apparently supports our initial data revealing the potential for over-expressed kindlin to suppress integrin activation as well as to cooperate with talin to activate integrins [128].

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In addition to direct competition with talin binding, regulation of integrin trafficking and recycling can modulate global integrin activation and function [189,198]. These processes are important for cell motility, but are likely important for adhesion maintenance and remodeling in constitutively adherent cells. We have observed integrin exocytosis at focal adhesions potentially providing new material for growing adhesions [199], but internalization of integrins by clathrindependent or non-clathrin-dependent means also provides a mechanism to turn over adhesions [189,198]. Such remodeling occurs during cell division, for example, where adhesion complexes are modified throughout cell-cycle progression to control cell rounding and subsequent division in a CDK1-dependent manner [200]. Internalized integrin may then be trafficked to lysosomes for degradation, or recycled back to adhesions at the cell surface to support anchorage and/or cell motility through interaction with various binding partners [199,201]. 4. Epithelial integrins Fibroblasts derived from knockout mice lacking specific integrin subunits, or integrin-associated proteins, have been an important for elucidation of integrin function [202] and, in recent years, the role of integrins in fibroblasts has received increasing attention in the context of fibrosis,

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Journal Pre-proof wound healing and tumor stroma interactions [70]. However, integrins also play essential roles in epithelial cells. Integrin-mediated cell-matrix interactions in epithelial cells are critical for anchoring cells to the basement membrane, and mediating the signaling that maintains cell survival [190,203]. As a means to restrict aberrant cell growth, non-cancerous epithelial cells are ‘anchorage dependent’, and undergo a form of apoptosis known as anoikis upon loss of cellmatrix interactions [204]. Thus, integrin-mediated cell-matrix interactions in epithelial cells are essential for survival of non-malignant epithelial cells.

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Integrins in epithelial cells have been demonstrated to be essential components of the signaling that serves to both establish the basement membrane and control epithelial adhesion to the basement membrane. In vitro and in vivo, the collagen and laminin-rich basement membrane is secreted in a polarized manner by epithelial cells in tandem with other cell types [205]. The integrins most highly expressed in epithelial cells are those which recognize key constituents of the basement membrane such as laminin (α3β1, α6β1, and α6β4) [203]. Other classes of integrins can also be found in epithelial cells, such as the vitronectin receptor αvβ5, and the fibronectin receptors α5β1 and αvβ6 [203]. The global and local expression of each integrin subunit is tightly regulated, and tends to be altered during epithelial repair in response to injury to concentrate integrins at the site of wounding [206].

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Although multiple collagen- and laminin-binding integrins are found to be abundant in epithelial cells, the function of each integrin in epithelial cell adhesion is non-redundant. For example, in mouse kidney collecting duct epithelial cells, deletion of α3β1 has the most severe effect on collecting duct cell adhesion to laminin and collecting duct cell proliferation, in comparison to α6β1 [207]. However, deletion of both α3 and α6 subunits worsens the phenotype, suggesting a synergistic effect for α3β1 and α6β1 with regards to laminin binding and cell adhesion [208].

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4.1 Hemidesmosomes and focal adhesions

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In certain classes of epithelial cells, integrin linkages to the ECM are mediated by hemidesmosomes, specialized multi-protein complexes including α6β4 integrins, that link intermediate filaments in epithelial cells to the basement membrane (Fig. 6A) [209,210]. However, epithelial integrins are not exclusively restricted to hemidesmosomes; integrins can also be found on the basal surface (α3β1 and α6β4) and apical surface (α2β1and α3β1), and can potentially mediate additional cell-matrix and cell-cell interactions [203]. In addition to hemidesmosomes, integrin-containing focal adhesions also serve as a second major linkage between epithelial cells and the underlying basement membrane (Fig. 6B) [210]. In contrast to hemidesmosomes, focal adhesions in epithelial cells serve to connect the actin cytoskeleton to the basement membrane through indirect integrin-actin connections [210].

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Figure 6: Hemidesmosomes vs. focal adhesions. (A) Hemidesmosomes connect integrins to the intermediate filament cytoskeleton, while (B) focal adhesions link integrins to the F-actin cytoskeleton, both directly and indirectly. 4.2 Cell adhesion during mitosis

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Given the importance of anchorage-dependent growth in non-malignant epithelial cells, maintenance of cell adhesion throughout a cell’s lifetime is essential for survival. However, during mitosis, disassembly of classical focal adhesions are observed, in addition to cell rounding [211]. How can a cell divide while maintaining anchorage? ‘Reticular adhesions’ (or clathrin plaques) are a recently recognized class of cell-matrix adhesions that are important for maintaining attachment between a cell and the underlying matrix during interphase and mitosis in epithelial cells and other cell types [211,212]. Interestingly, reticular adhesions are devoid of canonical focal adhesion components, such as talin and F-actin [211,212]. Analysis of these reticular adhesions suggests that they are rich in αvβ5 integrin, PI(4,5)P-binding intracellular proteins and components of the endocytic machinery [211–213]. Given the vast diversity in underlying substrate that cell can adhere to, it is likely that other integrin heterodimers may have roles in mitosis and reticular adhesions or similar mitotic structures.

4.3 The role of integrins in establishing epithelial cell polarity The polarized localization of integrins at the basal surface of the epithelium is important for establishing local asymmetry that is ultimately essential for higher-order tissue architecture. The importance of integrins in the establishment and maintenance of epithelial polarity in vitro has been well established in several functional studies. Knockout of β1 integrins in mammary epithelial cells has been shown to disrupt lumen formation and apical polarity, in a manner that is dependent on downstream signaling effectors such as ILK [214]. Notably, the vinculin-talin 20

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signaling axis is also important for establishment of the mammary epithelial cell phenotype by controlling the expression of milk protein genes, suggesting that signaling downstream of integrins can influence epithelial cell differentiation [215]. Similarly, addition of a functionblocking antibody against β1 integrin to MDCK cells in 3-D culture leads to defects in establishment of polarity and laminin organization in a manner that is dependent on signaling by Rac1 [216]. Rac1 signaling downstream of integrins is also critical for polarized laminin secretion and assembly at the basal surface, triggered by spatial and temporal cues from the ECM trigged by integrin-ECM engagement [217]. Overall, the integrins play an important role in establishment of cell polarity that is facilitated by their basally-polarized localization and roles in basement membrane secretion and organization.

5. Concluding Remarks

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Acknowledgements

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Integrins are adhesion receptors that mediate communication between the extracellular and intracellular environments and are thus essential for global tissue architecture and multicellularity. The regulatory pathways underlying integrin activity and signaling are remarkably complex and we are only beginning to fully understand the mechanistic details of integrin regulation. Complex structural rearrangements in the integrin heterodimers directly regulate affinities for ligands, and the dependence of these structural transitions on cytoplasmic binding partners such as the activating proteins talin and kindlin, and the negative regulators ICAP-1α, filamin, and SHARPIN, adds an additional layer of control. The fine-tuned balance between integrin activation and inactivation allows integrins to control cell anchorage and adhesion, and influence processes such as cell adhesion during mitosis and control of cell polarity in epithelial cells. Future studies examining the intracellular adhesome and signaling networks directly downstream of integrins will shed light into how these functions are controlled at an intracellular signaling level.

The authors would like to thank members of the Calderwood lab for helpful discussions and insights.

Funding The authors are supported by the National Institutes of Health grants R01-NS111980, R01NS093704 and R03TR002912 to D.A.C and a National Science Foundation Graduate Research Fellowship (DGE1122492) to Y.A.K.

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Highlights – Kadry et al. 2019 • Integrins couple the extracellular environment to intracellular signals • Thermodynamic regulation of integrin conformation controls integrin activity • Integrin activity is tightly regulated by binding partners (e.g. talin and kindlin) • Integrins play essential roles in epithelial cell adhesion and polarity

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